During development, spermatozoa exhibit a remarkable capability to respond to environmental cues, which dramatically alter their cell surfaces, transduction cascades, and motility. Spermatozoan development roughly can be divided into four phases, i.e., spermatogenesis, spermiation, ejaculation, and egg interaction. During spermatogenesis, spermatogonia in juxtaposition with Sertoli cells proliferate and differentiate into spermatozoa, and are immotile. In spermiation, sperm are discharged from the testis and acquire the potential for motility as they reach the vas deferens. Upon ejaculation, sperm motility is initiated. Further changes in functional competence and motility occur upon interaction of sperm with the follicular fluid and egg (i.e., in species with internal fertilization), or with water and egg (i.e., in species with external fertilization).
Specific cellular effectors present on the surface of sperm have been implicated at each developmental stage. However, relatively little is known about these receptors that transduce the environmental signals. In comparison, some of the molecular mediators of sperm activation and motility, which interact with the sperm receptors, have been identified.
As reviewed by Ronnett et al., Neuron, 15, 11-16 (1995), sperm developmental studies have revealed roles for environmental ionic composition, calcium, cyclic nucleotides such as cAMP and cGMP, protein modification such as protein phosphorylation and, potentially, protein carboxyl methylation, as well as other factors, at each sperm developmental stage. In particular, these environmental factors, and the cellular receptors with which they interact, have been implicated in the acquisition or exhibition of motility in sperm or sperm precursor cells. Moreover, receptor binding of unidentified chemoattractants appears to affect sperm chemotaxis (or the response of motile cells to a chemical gradient resulting in modulation of the direction of travel so as to approach an attractant or move away from a repellent) and sperm chemokinesis (or a change in swimming speed in response to a chemical stimulus). Both chemotaxis and chemokinesis ostensibly are important in the mammalian sperm-egg interaction.
While relatively little is known about sperm receptors in complex species such as mammals, putative egg peptide receptors have been identified in the sea urchin, Arbacia punctulata. Specifically, the sea urchin demonstrates sperm chemotaxis and chemokinesis toward peptides isolated from the jelly layer of the egg. Receptor cross-linking studies reveal binding of the egg peptides to a 77 kD cell-surface receptor, which is related to the LDL receptor, and to a 160 kD cell-surface receptor, which has been shown to be a membrane guanylyl cyclase.
This discovery of a classic component of a plasma membrane-associated signal transduction pathway in sperm provides a molecular basis for the increase in cGMP observed in sea urchin chemotaxis and chemokinesis studies. Moreover, the elucidation of components involved in sperm chemotaxis and chemokinesis, which typically characterize plasma membrane receptor signal transduction pathways, is interesting inasmuch as many of the same signal transduction pathways are activated in olfaction. This suggests that these systems (i.e., the olfactory system, and the sperm receptors and the signal transduction elements with which they interact in transducing environmental signals) may share relevant molecular components.
In terms of the overall design of the peripheral olfactory system, the olfactory cilia are the site of odor recognition and signal transduction. The specialized cilia are found on the dendritic knob in which the bipolar olfactory receptor neuron (i.e., the ORN) terminates at the surface of the neuroepithelium. The ORN is the primary sensory unit of the olfactory system and responds to odorant stimulation with a graded receptor potential, which results in an action potential, if the threshold is attained. The odorants activate adenylyl cyclase to increase cAMP in cilia. Activation occurs only in the presence of GTP, which suggests that the cascade is initiated when the odorants bind to seven transmembrane-spanning domain receptors coupled to guanine nucleotide binding proteins (i.e., G proteins). At least eighteen members of the multigene family that encodes the seven transmembrane domain proteins whose expression is limited to the olfactory epithelium have been identified (Buck et al., Cell, 65, 175-87 (1991)). Moreover, other signaling pathways appear to be activated by odorants to permit fine-tuning of the olfactory response. For instance, receptor-mediated stimulation of phospholipase C (PLC) generates inositol 1,4,5-triphosphate (InsP.sub.3), which binds to an InsP.sub.3 receptor located in the endoplasmic reticulum, resulting in a release of calcium from intracellular stores.
As regarding further similarities between the olfactory system and the sperm signal transduction system, more recent studies confirm that mediators of olfactory desensitization localize to the testis. Specifically, .beta.-ARK-2 and .beta.-arrestin-2, which are isoforms of chemosensory signalling desensitization proteins that are highly specific to olfactory receptor neurons, also are expressed in testis and round spermatids (Dawson et al., Science, 259, 825-829 (1993)). In contrast, .beta.-ARK-1 and .beta.-arrestin-1, the most ubiquitous isoforms of these proteins, are absent from both testis (i.e., round spermatids) and olfactory receptor neurons (Dawson et al. (1993), supra). These results confirm that highly specific isoforms of chemosensory signaling desensitization proteins are shared between olfactory receptor neurons and mature sperm.
Other studies similarly confirm the existence of odorant receptors in the male germ line, particularly in spermatocytes and spermatids (Parmentier et al., Nature, 355, 453-456 (1992); and Vanderhaeghen et al., J. Cell Biology, 123(6) 1441-1452 (1993)). Moreover, subcellular localization studies demonstrate that olfactory receptors and signal transduction proteins colocalize to the midpiece of mammalian sperm (Walensky et al., Molecular Medicine, 1(2), 130-141 (1995)). The respiratory center of the sperm is located in the tail midpiece, in close proximity to mitochondria and microtubule origins, and, thus, this region likely contains important signal transduction proteins. Accordingly, these results are consistent with a role for odorant receptors in transducing chemotactic signals in sperm.
Clearly, there is a need for a more thorough understanding of the response of spermatozoa to environmental cues, as effected by sperm receptors. Cloning of these sperm receptors can facilitate the understanding of the mechanism of sperm chemosensing by allowing production and further study of the receptors, as well as isolation of additional sperm receptors. Such production will enable, inter alia, identification of those ligands that interact with the receptors. This is advantageous, inasmuch as the ligands that bind to sperm can be difficult to isolate and analyze (e.g., due to their presence in very small amounts, or in gradients, which can vary only slightly, to effect a response). Moreover, it will allow manipulation of the sperm via manipulation of the receptor, e.g., as a means of contraception, and will provide a means of detecting autoimmune infertility.
Thus, there remains a need for a source of sperm receptors, to facilitate the further study of these receptors, as well as to identify additional receptors and ligands that bind to the receptors. There also remains a need for ways to alter the behavior of sperm, e.g., for use in contraception, and a need for a way to detect autoimmune infertility. Furthermore, there remains a need for a method to obtain a nucleic acid molecule that comprises a sequence that encodes a sperm receptor. The present invention provides nucleic acids encoding sperm receptors, thus remedying these needs. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention set forth herein.